Analysis of Differentially Expressed Proteins in Colorectal Cancer Using Hydroxyapatite Column and SDS-PAGE

Abstract

Limitation on two dimensional (2D) gel electrophoresis technique causes some proteins to be under presented, especially the extreme acidic, basic, or membrane proteins. To overcome the limitation of 2D electrophoresis, an analysis method was developed for identification of differentially expressed proteins in normal and cancerous colonic tissues using self-pack hydroxyapatite (HA) column. Normal and cancerous colon tissues were homogenized and proteins were extracted using sodium phosphate buffer at pH 6.8. Protein concentration was determined and the proteins were loaded unto the HA column. HA column reduced the complexity of proteins mixture by fractionating the proteins according to their ionic strength. Further protein separation was accomplished by a simple and cost effective sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. The protein bands were subjected to in-gel digestion and protein analysis was performed using electrospray ionization (ESI) ion trap mass spectrometer. There were 17 upregulated proteins and seven downregulated proteins detected with significant differential expression. Some of these proteins were low abundant proteins or proteins with extreme pH that were usually under presented in 2D gel analysis. We have identified brain mitochondrial carrier protein 1, T-cell surface glycoprotein CD1a, SOSS complex subunit B2, and Protein Jade 1 which were previously not detected in 2D gel analysis method.

Structure, function and regulation of jade family PHD finger 1 (JADE1)

“…The results of these studies do not always reconcile perhaps due to the fact that they were generated predominantly from the histochemical analysis of tumor specimens using JADE1 antibody with uncharacterized specificities towards JADE1 in general, and JADE1S or JADE1L in particular 63, 64 . Results of a study using in silico microarray algorithm analysis show that PHF17 mRNA may play a role in the development of pancreatic cancer 65 .…”

Genome-wide association study of colorectal cancer in Hispanics

“…Subsequently, this SNP was replicated in one recent meta-analysis of Europeans and East Asians but not in a second ( 20 , 32 ). Although the most proximal gene is not necessarily the most functionally relevant, it is worth noting that single-stranded DNA binding protein NABP1’s potential link to cancer development has been suggested in relation to the maintenance of genomic instability as a component of the sensor of ssDNA (SOSS) complex; also, NAPB1 was found in a proteomic screen of differentially expressed proteins in CRCs ( 64 , 65 ). Here, we did not replicate this index SNP, but we did observe that rs12474044, a SNP in LD with the index that is approximately 10kb away ( r 2 AMR = 0.12; r 2 EUR = 0.16), was associated with risk of CRC at a nominal level of statistical significance.…”

Spontaneous Unexplained Preterm Labor with Intact Membrane: Finding Protein Biomarkers through Placenta Proteome

“…It is possible that limitations exist in the 2D-GE technology. This is due to the presence of proteins that are extremely acidic and basic, hydrophobic, large molecular weights, and low copy numbers [16]. Furthermore, limitations were observed when identifying and quantifying proteins as the protein spots need to be excised out from the 2D gels and digested with proteases.…”